Prostate cancer is the second most common cause of cancer related death and will kill an estimated 37,000 people this year alone. The prostate gland, which is found exclusively in male mammals, produces several regulatory peptides. The prostate gland comprises stroma and epithelium cells, the latter group consisting of columnar secretory cells and basal non-secretory cells. A proliferation of these basal cells, as well as stroma cells gives rise to benign prostatic hyperplasia (BPH) which is one common prostate disease. Another common prostate disease is prostatic adenocarcinoma (CaP), the most common of the fatal pathophysiological prostate cancers. Prostatic adenocarcinoma involves a malignant transformation of epithelial cells in the peripheral region of the prostate gland. Prostatic adenocarcinoma and benign prostatic hyperplasia are two common prostate diseases which have a high rate of incidence in the aging human male population. Approximately one out of every four males above the age of 55 suffers from a prostate disease of some form or another.
To date, various substances that are synthesized and secreted by normal, benign and cancerous prostates are used as tumor markers to gain an understanding of the pathogenesis of the various prostate diseases and in the diagnosis of prostate disease. The three predominant proteins or peptides secreted by a normal prostate gland are Prostatic Acid Phosphatase (PAP), Prostate Specific Antigen (PSA) and prostatic inhibin (PIP) also known as human seminal plasma inhibin (HSPI). Both PSA and PAP have been studied as tumour markers in the detection of prostate disease but since both exhibit elevated levels in prostates having benign prostatic hyperplasia (BPM) neither marker is specific and therefore are of limited use.
Despite the available knowledge, little is known about the genetic basis underlying the prostate cancer disease and the androgen-regulated genes that may be involved with its progression. Although androgens have been known to play a major role in the biology of prostate cancer. However, the full complexity of the hormonal regulation has not been completely covered and more androgen related processes are being elucidated. Many of these processes involve several molecules associated in prostate cancer that remain elusive. In addition, there may be several known molecules that have not yet been associated with the pathogenesis of the disease. Accordingly, a need exists for identifying unknown molecules that may be involved in prostate cancer and the genes encoding them. A need also exists for identifying known molecules that have not yet been implicated in the pathogenesis of prostate cancer, particularly those that can serve as targets for the diagnosis, prevention, and treatment of prostate cancer.
The invention is based, in part, on the discovery of a number of genes which are androgen-inducible in androgen-dependent prostate cancer cells (e.g., LNCaP cells). These genes serve as markers suitable for detection, diagnosis and prognosis of prostate disorders. This invention provides methods and screening assays for the detection and diagnosis of prostate cancer. The primary screening assays detect an alteration in the expression level of genes associated with prostate cancer. In particular, this invention provides for the use of immunophilins, such as FK-Binding Proteins (FKBPs), e.g., FKBP54, as genetic markers for the detection, diagnosis and prognosis of prostate disorders. Immunophilins are proteins that serve as receptors for the immunosuppressant drugs such as cyclosporin A (CsA), FK506, and rapamycin. Known classes of immunophilins include cyclophilins, and FKS506 binding proteins, such as FKBPs. Cyclosporin A binds to cyclophilin while FK506 and rapamycin bind to FKBP. These immunophilin-drug complexes interface with a variety of intracellular signal transduction systems. Immunophilins are known to have peptidyl-prolyl isomerase (PPIase) or rotamase enzyme activity. It has been determined that rotamase activity has a role in the catalyzation of the interconversion of the cis and trans isomer of immunophilin proteins.
FKBP54 is a member of the immunophilin family and has been associated with the progesterone receptor complex as described by Smith et al. (1993) J. Biol. Chem. 268: 18365-18371. The invention provides for use of immunophilins, e.g., FKBP54, that are up-regulated (increased MRNA and protein expression/activated/agonized) or down-regulated (decreased mRNA and protein expression/suppressed/antagonized) in the presence of androgens.
Using gene cluster analysis, the expression pattern of FKBP54 was found to be similar to that of prostate specific antigen (PSA), which has been used to diagnose prostate cancer patient. The present study described herein demonstrates the up-regulation of FKBP54 in the presence of androgen and can be used as a marker for the detection, diagnosis and prognosis of prostate disorders. In addition, quantitative PCR was used to confirm gene expression of the target marker. The transcription level of FKBP54 was found to be regulated by androgen, demonstrating a time dependent increase in transcription. Western blot analysis of the expressed FKBP54 protein further confirmed the time dependent increase in expression levels in the presence of androgen. The presence of FKBP54 in solid tumors was also demonstrated. Furthermore, transient cotransfection studies in COS cells showed that androgen receptor activation was enhanced by FKBP54.
In one embodiment, the invention provides a method of assessing whether a subject is afflicted with prostate cancer, by comparing the level of expression of the FK-binding proteins, e.g., FKBP54 marker in a sample from a subject, to the normal level of expression of the marker in a control sample, where a significant difference between the level of expression of the marker in the sample from the subject and the normal level is an indication that the subject is afflicted with prostate cancer. In a preferred embodiment, the marker corresponds to a transcribed polynucleotide or portion thereof, where the polynucleotide includes the marker. In a particularly preferred embodiment, the level of expression of the marker in the sample differs from the normal level of expression of the marker in a subject not afflicted with prostate cancer by a factor of at least two, and in an even more preferred embodiment, the expression levels differ by a factor of at least three. In another preferred embodiment, the marker is not significantly expressed in non-prostate cancer cells.
In another preferred embodiment, the sample includes cells obtained from the subject. In another preferred embodiment, the level of expression of the marker in the sample is assessed by detecting the presence in the sample of a protein corresponding to the marker. In a particularly preferred embodiment, the presence of the protein is detected using a reagent which specifically binds with the protein. In an even more preferred embodiment, the reagent is selected from the group of reagents including an antibody, an antibody derivative, and an antibody fragment. In another preferred embodiment, the level of expression of the marker in the sample is assessed by detecting the presence in the sample of a transcribed polynucleotide or portion thereof, where the transcribed polynucleotide includes the marker. In a particularly preferred embodiment, the transcribed polynucleotide is an mRNA or a cDNA. In another particularly preferred embodiment, the step of detecting further comprises amplifying the transcribed polynucleotide.
In yet another preferred embodiment, the level of expression of the FKBP marker, e.g., FKBP54 marker in the sample is assessed by detecting the presence in the sample of a transcribed polynucleotide which anneals with the marker or anneals with a portion of a polynucleotide under stringent hybridization conditions, where the polynucleotide includes the marker. The level of expression of the marker is significantly altered, relative to the corresponding normal levels of expression the marker, is an indication that the subject is afflicted with prostate cancer.
In another embodiment, the invention provides a method for monitoring the progression of prostate cancer in a subject, including detecting in a subject sample at a first point in time the expression of the FKBP marker, e.g., FKBP54 marker, repeating this detection step at a subsequent point in time, and comparing the level of expression detected in the two detection steps, and monitoring the progression of prostate cancer in the subject using this information. In another preferred embodiment, the marker corresponds to a transcribed polynucleotide or portion thereof, where the polynucleotide includes the marker. In another preferred embodiment, the sample includes cells obtained from the subject. In a particularly preferred embodiment, the cells are collected from skin or blood tissue.
In another embodiment, the invention provides a method of assessing the efficacy of a test compound for inhibiting prostate cancer in a subject, including comparing expression of the FKBP54 marker in a first sample obtained from the subject which is exposed to or maintained in the presence of the test compound, to expression of the FKBP marker, e.g., FKBP54 marker in a second sample obtained from the subject, where the second sample is not exposed to the test compound, where a significantly lower level of expression of the marker in the first sample relative to that in the second sample is an indication that the test compound is efficacious for inhibiting prostate cancer in the subject. In a preferred embodiment, the first and second samples are portions of a single sample obtained from the subject. In another preferred embodiment, the first and second samples are portions of pooled samples obtained from the subject.
In another embodiment, the invention provides a method of assessing the efficacy of a therapy for inhibiting prostate cancer in a subject, the method including comparing expression of the FKBP marker, e.g., FKBP54 marker in the first sample obtained from the subject prior to providing at least a portion of the therapy to the subject, to expression of the marker in a second sample obtained form the subject following provision of the portion of the therapy, where a significantly lower level of expression of the marker in the second sample relative to the first sample is an indication that the therapy is efficacious for inhibiting prostate cancer in the subject.
In another embodiment, the invention provides a method of selecting a composition for inhibiting prostate cancer in a subject, the method including obtaining a sample including cells from a subject, separately maintaining aliquots of the sample in the presence of a plurality of test compositions, comparing expression of the FKBP marker, e.g., FKBP54 marker in each of the aliquots, and selecting one of the test compositions which induces a lower level of expression of the FKBP marker, e.g., FKBP54 marker in the aliquot containing that test composition, relative to other test compositions.
In another embodiment, the invention provides a method of inhibiting prostate cancer in a subject, including obtaining a sample including cells from a subject, separately maintaining aliquots of the sample in the presence of a plurality of test compositions, comparing expression of the FKBP marker, e.g., FKBP54 marker in each of the aliquots, and administering to the subject at least one of the test compositions which induces a lower level of expression of the FKBP marker, e.g., FKBP54 marker in the aliquot containing that test composition, relative to other test compositions.
In another embodiment, the invention provides a method of assessing the potential of a test compound to trigger prostate cancer in a cell, including maintaining separate aliquots of cells in the presence and absence of the test compound, and comparing expression of the FKBP marker, e.g., FKBP54 marker in each of the aliquots, where a significantly enhanced level of expression of the FKBP marker, e.g., FKBP54 marker in the aliquot maintained in the presence of the test compound, relative to the aliquot maintained in the absence of the test compound, is an indication that the test compound possesses the potential for triggering prostate cancer in a cell.
In another embodiment, the invention provides a method of treating a subject afflicted with prostate cancer, including providing to cells of the subject afflicted with prostate cancer a protein corresponding to the FKBP marker, e.g., FKBP54 marker. In a preferred embodiment, the protein is provided to the cells by providing a vector including a polynucleotide encoding the FKBP protein, e.g., FKBP54 protein to the cells.
In another embodiment, the invention provides a method of treating a subject afflicted with prostate cancer an antisense oligonucleotide complementary to a polynucleotide corresponding to the FKBP marker, e.g., FKBP54 marker.
In another embodiment, the invention provides a method of inhibiting prostate cancer in a subject at risk for developing prostate cancer, including inhibiting expression of a gene corresponding to the FKBP marker, e.g., FKBP54 marker.
Other features and advantages of the invention will be apparent from the following detailed description and claims.